LINEAGE TRACING OF p16INK4A REVEALS HETEROGENEOUS NATURE OF TUBULAR AGING IN THE KIDNEY

Senescent cells, characterized by cell cycle arrest and senescence-associated secretory phenotypes, play key roles in tissue aging and repair. p16INK4a is an established marker of cellular senescence, yet its detection in vivo remains challenging. Most studies on cellular senescence have relied on fibroblast cultures, leaving in vivo behavior of epithelial senescent cells unexplored. This study reveals the distribution of p16INK4a-positive senescent cells in the kidney, focusing particularly on their response to aging and injury.

We used a tamoxifen-inducible lineage-tracing system with p16CreERT2:R26-tdTomato mice to label p16INK4a-expressing cells. Kidney tissues were analyzed from aged mice (2-year-old), gonadectomized young mice, and Klotho-/- progeria mice, focusing on proximal tubules (PTs). CUBIC clearing enabled 3D visualization of tdTomato+ (tdT+) cell distribution within the nephron. Proximal tubular cells (PTCs) were isolated from p16CreERT2:R26-tdTomato mice by magnetic-activated cell sorting (MACS), followed by primary culture and DNA damage and proliferation assays. After tamoxifen labeling, unilateral ischemia-reperfusion injury (IRI) was induced, and kidneys were examined during acute and chronic phases. Photo-isolation chemistry (PIC) RNA-seq was performed using two classification strategies for PTCs based on tdT expression.

Approximately 70% of tdT+ cells were identified in PTs across sex and age groups, with aged females showing higher fractions. Tissue clearing revealed tdT+ cell clusters within PTs. In both sexes, gonadectomy increased tdT+ PTCs, indicating gonadal dysfunction may enhance p16INK4a expression. Klotho-/- progeroid mice showed increased tdT+ PTCs, consistent with enhanced p16INK4a expression during premature senescence. In vitro, tdT+ PTCs exhibited more γH2AX foci after DNA damage and slower proliferation, indicating stronger DNA damage response and reduced proliferation. In the IRI model, tdT⁺ PTCs decreased in VCAM1+ failed repair PTs at day 14. Consistently, quantitative imaging 3 min after reperfusion showed that tdT⁺ PTCs were significantly more detached from the tubular basement membrane, suggesting impaired adhesion. To understand their accumulation in aged female mouse kidneys, we performed PIC RNA-seq comparing gene expression of tdT+ PTCs, tdT- PTCs within the same tubule with tdT+ PTC (Neighbor PTC), and tdT- PTCs from tdT- PTs (Control PTC). tdT+ PTCs showed signatures of DNA damage and reduced cell adhesion, while both tdT+ and Neighbor PTCs exhibited upregulation of genes related to mitochondrial dysfunction and ER stress, supported by immunostaining and electron microscopy. These changes suggest stress-associated alterations occur at the tubule level. Additional PIC RNA-seq revealed distinct transcriptomic profiles between tdT+ and tdT- PTs indicating heterogeneous aging progression among nephrons.

Tamoxifen-inducible lineage tracing of p16INK4a successfully visualized tubular aging at the cellular level. p16INK4a+ PTCs accumulated in aged female mouse kidneys, exhibiting DNA damage, impaired proliferation, and were more prone to detachment upon injury. Spatial transcriptomics revealed nephron-dependent heterogeneity in PT stress responses, providing insights into kidney aging dynamics. This work was first presented at ASN Kidney Week 2023, and re-submission is permitted by ASN.